dNTPs/PCR

[PrinceAli2786]

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Hey, can someone plz simplify for me the whole dNTP and PCR concept, I read over it but just don't fully understand it w/out confusing myself and how will this be tested on the MCAT really?

Can you plz give like a fake example to go along with the explanation I'd really appreciate it, since I've heard a lot of GENETICS is tested now.

Thanks!😎

 

[travelbug73]

I don't know that you require the whole PCR concept for the MCAT. But here it is since you asked🙂

PCR = Polymerase Chain Reaction. In simple terms it is a logarithmic increase of your starting material which is DNA.

The ingredients you need for a successful PCR are:

1) template DNA

2) primers: equal to a diving board, the DNA needs an anchor to start replicating. In the cell primers are short RNA pieces while in the test tube they are short single strand DNA pieces. You need two of these, one for the top strand and another for the bottom to replicate your region of interest.

3) Magnesium Chloride: needed for Polymerase activity and also for a lot of other things

4) dNTPs: Ok, you have the primers but if you don't have more nucleotides to pull from, your primers cannot do a thing. Basically, dNTPs (dATP, dCTP, dGTP, and dTTP) are a pool of nucleotides that are present in the reaction mix. Every time the Polymerase sees a particular nucleotide on the parent DNA strand, it will go looking for the complementary nucleotide in the pool. So, let's say, the Polymerase came across dATP on the original strand of DNA, it will bring a dTTP from the pool to complementarily base pair with the dATP.
5) 10X buffer: mainly to stabilize polymerase

6) DNA Polymerase: Enzyme needed for replication.

During PCR you are amplifying DNA. Let's say you have 1 copy of DNA, you have millions of copies at the end of PCR. How do you get from one copy to millions of copies?

You add your template DNA

You denature the DNA to make it into two strands, done at 95C

At about 55-60C the primers will bind (anneal) in a complementary fashion to the top or the bottom strand

At about 72C you extend the primers by adding nucleotides and where are these nucleotides coming from? Voila! dNTPs.

This goes on for about 35-45 cycles at the end of which you have your amplified product. By the time you reach the end many reagents become limiting and so your PCR reaction will have plateaued.

Too much information perhaps but...

 

[PrinceAli2786]

Thanks so much for the info/reply!👍

So, its basically a way to make copies of DNA in vitro? rather than normally in vivo? Or is it used to just identify the parent DNA strand?😕


But how would a problem be presented? Any idea?

Like will it show a data printout of the reaction, like that thing w/ the horizontal lines going from bottom to top? at either the dATP, dCTP, dGTP, dTTP, etc? And just ask what is the parent strand?

Or will it be more complicated and if so any idea how?

 

[iA-MD2013]

Thanks so much for the info/reply!👍

So, its basically a way to make copies of DNA in vitro? rather than normally in vivo? Or is it used to just identify the parent DNA strand?😕


But how would a problem be presented? Any idea?

Like will it show a data printout of the reaction, like that thing w/ the horizontal lines going from bottom to top? at either the dATP, dCTP, dGTP, dTTP, etc? And just ask what is the parent strand?

Or will it be more complicated and if so any idea how?

I don't know how it would be asked (haven't taken the test yet) but the questions I've seen in practice tests are very simple. For example, one practice test asked why you can't use human DNA polymerase in PCR (it would denature at the high temp needed to separate the strands of DNA)
PCR is used to amplify the amount of DNA in vitro. If you have a gene in some DNA that you want/need more of, you use PCR to make more of it.

 

[PrinceAli2786]

I don't know how it would be asked (haven't taken the test yet) but the questions I've seen in practice tests are very simple. For example, one practice test asked why you can't use human DNA polymerase in PCR (it would denature at the high temp needed to separate the strands of DNA)
PCR is used to amplify the amount of DNA in vitro. If you have a gene in some DNA that you want/need more of, you use PCR to make more of it.

makes sense🙂, k cool thanks for the help/info too👍

 

[travelbug73]

Thanks so much for the info/reply!👍

So, its basically a way to make copies of DNA in vitro? rather than normally in vivo? Or is it used to just identify the parent DNA strand?😕


But how would a problem be presented? Any idea?

Like will it show a data printout of the reaction, like that thing w/ the horizontal lines going from bottom to top? at either the dATP, dCTP, dGTP, dTTP, etc? And just ask what is the parent strand?

Or will it be more complicated and if so any idea how?

I don't think you need to focus so much on PCR as you would probably want to on the basic structure of nucleic acids and the central dogma: DNA to RNA to protein. You also need to know the basics of reverse transcription, complementary base pairing, nucleic acid synthesis direction (always 5' to 3').
There might be a question on RE digests in patient vs. normal that asks you to determine who has a mutation and from which parent it has possibly been inherited. But, all of this information should be given in the passage.
I don't know what kind of specific PCR questions they could ask except after they have explained their experiment in the passage. For example they could ask (though I don't believe it is within the scope of MCAT) why a particular experiment failed. And in that experiment they could have left out one of the ingredients such as dNTP or primer or something like that.
PCR has many applications. The bottom line is to identify the sequence of DNA and determine if it has a mutation or if it is related to something (microbiology) or someone else (paternity testing, criminal identity) etc.

 

[PrinceAli2786]

I don't think you need to focus so much on PCR as you would probably want to on the basic structure of nucleic acids and the central dogma: DNA to RNA to protein. You also need to know the basics of reverse transcription, complementary base pairing, nucleic acid synthesis direction (always 5' to 3').
There might be a question on RE digests in patient vs. normal that asks you to determine who has a mutation and from which parent it has possibly been inherited. But, all of this information should be given in the passage.
I don't know what kind of specific PCR questions they could ask except after they have explained their experiment in the passage. For example they could ask (though I don't believe it is within the scope of MCAT) why a particular experiment failed. And in that experiment they could have left out one of the ingredients such as dNTP or primer or something like that.
PCR has many applications. The bottom line is to identify the sequence of DNA and determine if it has a mutation or if it is related to something (microbiology) or someone else (paternity testing, criminal identity) etc.

k got it, thanks! Only reason I got worried was I took a KAPLAN subject test on genetics and it had a hard PCR passage on it. Thanks a lot for the help you guys👍